Abstract

Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS.

Figure 1

(A) Schematic of the Rag2 protein showing the core and non-core regions and enhancement of the C-terminal non-core region that includes the acidic hinge region, the PHD, and the noncanonical NLS overlapping the phosphorylation site at residue T490 (thin black vertical bar) and the cationic region between residues 499–508. Thick black vertical bars indicate residues mutated in OS/SCID patients; mutations analyzed in this study are in bold. Anchor residues forming the noncanonical PHD finger are indicated, and related interactions with Zinc ions (Zn1 and Zn2) are represented by gray bars; L1 and L2 indicate segments forming loops between pairs of zinc-coordinating residues, as previously described (18). C and H represent Zn+2 coordinating cysteine and histidine residues present in the Rag2 PHD domain. (B) A mouse Rag2–/– pro-B cell line was retrovirally transduced and selected to express constructs of interest: empty vector (Mock), FNT tag, FNT-tagged R2CR, C terminus of Rag2 (R2CT), R2FL, or full-length T-B-SCID/OS Rag2 mutants. Genomic DNA was harvested from the above cell lines, and Southern Blot analysis was performed on PCR-amplified products of IgH D-to-J, VH7183-to-DJ, VHQ52-to-DJ, and Vκ-to-Jκ rearrangements, as indicated to the left of each panel. Numbers of the left side of the figure (1 to 5) represent different recombination products. Triangles at the top of panels indicate PCR amplification of 400 ng, 200 ng, and 25 ng of DNA template. A fragment of Rag1 gene was amplified as a loading control. For each type of rearrangement, samples were loaded on the same gel, and detection intensity was identical. PCR from nontransduced cells was also used as control. The results shown are representative of 2 independent experiments. mRag2–/–, mouse Rag2–/–.